Method for detecting multispecific antibody light chain mispairing

ABSTRACT

Use of a limited digestion with a proteolytic enzyme of a multispecific antibody for the analysis of the multispecific antibody&#39;s light chain pairing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent Application No. PCT/EP2015/057164, having an international filing date of Apr. 1, 2015, the entire contents of which are incorporated herein by reference, and which claims benefit under 35 U.S.C. §119 to European Patent Application No. 14163165.5, filed on Apr. 2, 2014 and European Patent Application No. 15155361.7, filed on Feb. 17, 2015.

BACKGROUND OF THE INVENTION

Herein is reported a method for the determination of light chain mispairing in a multispecific antibody preparation based on limited proteolytic digestion and ESI-MS analysis. The method can be used for quality control or cell line selection.

In 1995 Yamaguchi et al. (J. Immunol. Meth. 181 (1995) 259-267) reported a method for proteolytic fragmentation with high specificity of mouse immunoglobulin G using lysyl endopeptidase, clostripain, metalloendopeptidase and V8 protease, whereby under the reaction conditions examined clostripain, lysyl endopeptidase, metalloendopeptidase, and V8 protease failed to cleave IgG1 and IgG1-CM selectively in the hinge region.

Matsuda et al. (FEBS Lett. 473 (2000) 349-357) reported a method for the preparation of Fc fragments using Lys-C for obtaining information of the Fc-associated glycoforms by MS for determining the pairing of oligosaccharides in the Fc region of immunoglobulin G.

Villanueva et al. (J. Mass Spectrom. 37 (2002) 974-984) reported a limited proteolysis methodology, based on the use of unspecific exoproteases coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), to map quickly secondary structure elements of a protein from both ends, the N- and C-termini.

In WO 2005/073732 a method of analysis of high molecular weight proteins, specifically, a reversed-phase LC/MS method of analysis of high molecular weight proteins including antibodies, is reported.

WO 2006/047340 relates to subjecting column isolated preparations of polypeptides with a reduction/oxidation reagent and a chaotropic agent and isolating the refolded active protein produced from said contacting. The active proteins can be high molecular weight proteins including Fc domain containing polypeptides or fragments.

Kleemann et al. (Anal. Chem. 80 (2008) 2001-2009) reported the characterization of IgG1 immunoglobulins and peptide-Fc fusion proteins by limited proteolysis in conjunction with LC-MS. Applying limited proteolysis using endoproteinase Lys-C resulted in the predominant cleavage C-terminal of this lysine residue.

SUMMARY OF THE INVENTION

It has been found that for the determination of light chain mispairing in a multispecific antibody limited proteolysis of the (multispecific) antibody has to be performed prior to MS analysis as the determination of light chain mispairing in a complete multispecific antibody is hampered by isobaric mass interference.

Thus, one aspect as reported herein is the use of a limited digestion with a proteolytic enzyme of a multispecific antibody for the analysis of the multispecific antibody's light chain pairing.

One aspect as reported herein is the use of a limited digestion with a proteolytic enzyme of a multispecific antibody for the determination of light chain mispairing in the multispecific antibody.

One aspect as reported herein is the use of a limited digestion with a proteolytic enzyme of a multispecific antibody produced by a recombinant mammalian cell for the selection of a multispecific antibody producing mammalian cell.

In one embodiment of all previous aspects the analysis/determination/selection is by using the result of a mass spectrometry of the digested antibody.

One aspect as reported herein is a method for the determination of the light chain pairing in a multispecific antibody comprising the following steps:

-   -   a) incubating a sample comprising the multispecific antibody         with a proteolytic enzyme for a limited time,     -   b) identifying the mass of the fragments obtained by the limited         proteolytic digestion in step a) by mass spectrometry, and     -   c) determining from the results in step b) the light chain         pairing of the multispecific antibody.

One aspect as reported herein is a method for the determination of light chain mispairing of a multispecific antibody comprising the following steps:

-   -   a) incubating a sample comprising the multispecific antibody         with a proteolytic enzyme for a limited time,     -   b) identifying the mass of the fragments obtained by the limited         proteolytic digestion in step a) by mass spectrometry, and     -   c) determining from the results in step b) the light chain         pairing of the multispecific antibody and thereby determining         light chain mispairing.

One aspect as reported herein is a method for the selection of a recombinant mammalian cell producing a multispecific antibody comprising the following steps:

-   -   a) individually incubating a sample comprising a multispecific         antibody produced by a clonal population of a recombinant         mammalian cell of a multitude of recombinant mammalian cells all         cells of the multitude producing the same multispecific antibody         with a proteolytic enzyme for a limited time,     -   b) identifying the mass of the fragments obtained by the limited         proteolytic digestion in step a) by mass spectrometry,     -   c) determining from the results in step b) the presence of light         chain mispairing of the multispecific antibody for each clonal         cell population, and     -   d) selecting based on the results in step c) a recombinant cell         producing a multispecific antibody.         In one embodiment of all aspects the proteolytic enzyme is         selected from the group consisting of Lys-C, plasmin, Asp-N,         Arg-C, Glu-C, Ides, pepsin and chymotrypsin.

In one embodiment the multispecific antibody is a bivalent antibody.

In one preferred embodiment of all aspects the proteolytic enzyme is Lys-C or plasmin.

In one embodiment the multispecific antibody is a tri- or tetravalent antibody. In one embodiment the multispecific antibody is a full length antibody comprising two light chains and two heavy chain whereby to the C-termini of the heavy chains a moiety independently of each other selected from the group consisting of no moiety, an Fv fragment, a Fab fragment, a scFv fragment and a scFab fragment is conjugated either directly or via a peptidic linker.

In case the antibody is a tetravalent antibody wherein also binding sites are attached to the C-termini of the heavy chains a double digestion with two proteolytic enzymes might be required.

In one embodiment the multispecific antibody is a trivalent or tetravalent multispecific antibody and the method comprises a double digestion by incubating with a mixture of two proteolytic enzymes.

In one embodiment the mixture of proteolytic enzymes is plasmin and Ides. In one embodiment the mixture of proteolytic enzymes is Lys-C and Ides.

In case of an effector silent antibody (L234A, L235A mutations) Ides does not cleave and pepsin has to be chosen.

In one embodiment the mixture of proteolytic enzymes is plasmin and pepsin. In one embodiment the mixture of proteolytic enzymes is Lys-C and pepsin.

In one preferred embodiment the multivalent antibody is a bispecific antibody.

In one embodiment of all aspects the incubating is for up to 60 minutes. In one embodiment of all aspects the incubating is for 20 to 60 minutes. In one embodiment of all aspects the incubating is for 35 to 45 minutes.

In one preferred embodiment of all aspects the incubating is for about 40 minutes.

In one embodiment of all aspects the multispecific antibody that is incubated with the proteolytic enzyme is a deglycosylated multispecific antibody.

In one embodiment of all aspects the weight ratio of antibody to enzyme is 1:150 to 1:500. In one embodiment of all aspects the weight ratio of antibody to enzyme is 1:200 to 1:400. In one embodiment of all aspects the weight ratio of antibody to enzyme is about 1:200.

In one embodiment of all aspects the multispecific antibody that is incubated with the proteolytic enzyme has a concentration of from 200 to 600 μg/mL.

In one embodiment of all aspects step b) is

-   -   b) desalting the incubation mixture of step a) and identifying         the mass of the fragments obtained by the limited proteolytic         digestion in step a) by mass spectrometry.

In one embodiment of all aspects the multispecific antibody is a monoclonal multispecific antibody.

In one embodiment of all aspects the multispecific antibody comprises at least two non-peptidically associated light chains.

In one embodiment of all aspects the multispecific antibody comprises at least three non-peptidically associated polypeptides.

In one embodiment of all aspects the mass spectrometry is an online determination.

In one embodiment of all aspects the mass spectrometry is an offline determination. In one embodiment the offline mass spectrometry is preceded by a desalting step.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. In contrast thereto reference to “one cell” does not include a plurality of such cells but is limited to a single (isolated) cell.

It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably.

Further it is also to be noted that the term “comprising” encompasses the term “consisting of” as limitation.

The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., at least bispecific antibodies) as long as these antibody structures are multispecific and comprise at least two non-peptidically bound light chains.

The term “non-peptidically bound” denotes that two polypeptides are not associated with each other by a peptide bond (formed between amino- and carboxy-groups of amino acids). Nevertheless these polypeptides can be associated by other covalent bonds such as disulfide bonds or non-covalently.

The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ respectively.

The term “Fc-region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc-regions and variant Fc-regions. In one embodiment, a human IgG heavy chain Fc-region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc-region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc-region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), NIH Publication 91-3242.

The terms “full length antibody”, “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt, T. J. et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

DETAILED DESCRIPTION OF THE INVENTION

Herein is reported a method for the determination of light chain mispairing in a multispecific antibody preparation based on limited proteolytic digestion and ESI-MS analysis. The method can be used for quality control or cell line selection.

It has been found that for the determination of light chain mispairing in a multispecific antibody limited proteolysis of the (multispecific) antibody has to be performed prior to MS analysis as the determination of light chain mispairing in a complete multispecific antibody is hampered by isobaric mass interference.

Thus, one aspect as reported herein is the use of a limited digestion with a proteolytic enzyme of a multispecific antibody for the analysis of the multispecific antibody's light chain pairing.

In a bivalent multispecific antibody, i.e. in a bivalent bispecific antibody, one light chain mispairing product is possible (see FIG. 1).

In one exemplary embodiment of the methods as reported herein two samples of a bivalent bispecific antibody has been digested with plasmin (EC 3.4.21.7). In all samples no uncleaved intact antibody was present after the digestion. In sample 1 approximately 20% of light-chain mispaired antibody is detectable (see FIG. 3) whereas in the second sample no light-chain mispaired antibody is detectable (see FIG. 4).

In one exemplary embodiment of the methods as reported herein a sample of a bivalent bispecific antibody has been limited digested with Lys-C. In the sample light-chain mispaired antibody is detectable (see FIG. 5).

If more than 2 light chains are present, e.g. in a trivalent or tetravalent multispecific antibody, more than one light chain mispairing product is possible (see FIG. 2). In a mass spectrometric analysis of the complete antibody it is only possible to determine the presence of a light chain mispaired product but not the stoichiometry and the orientation of a potential mispairing cannot be detected (see FIG. 6).

Different proteolytic enzymes can be chosen for this task:

-   -   Ides: cleaves below the hinge region (cannot be used in an         antibody with the L234A, L235A mutation)     -   plasmin: cleaves above the hinge region     -   pepsin: cleaves below the hinge region; results in a degraded         Fc-region     -   Lys-C: cleaves above the hinge region.

In one exemplary embodiment of the methods as reported herein a sample of a tetravalent bispecific antibody has been limited digested with Lys-C.

It has been found that for a tetravalent multispecific antibody the mispairing in the Fab region form from light chain 1 and the heavy chain 1 fragment can be detected better at an ISCID of 0 whereas the Fc-region fragment comprising the second light chain can be detected better at an ISCID of 90.

In order to remove the Fc-region a Protein A affinity chromatography can be used to separate the Fab fragments from the Fc-region after the enzymatic digestion. The Fab fragments are in the flow-through of the Protein A affinity chromatography.

Chimeric and Humanized Antibodies

In certain embodiments, an antibody analyzed with the methods as reported herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison, S. L. et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., in Almagro, J. C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and are further described, e.g., in Riechmann, I. et al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033; U.S. Pat. No. 5,821,337, U.S. Pat. No. 7,527,791, U.S. Pat. No. 6,982,321, and U.S. Pat. No. 7,087,409; Kashmiri, S. V. et al., Methods 36 (2005) 25-34 (describing specificity determining region (SDR) grafting); Padlan, E. A., Mol. Immunol. 28 (1991) 489-498 (describing “resurfacing”); Dall'Acqua, W. F. et al., Methods 36 (2005) 43-60 (describing “FR shuffling”); and Osbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims, M. J. et al., J. Immunol. 151 (1993) 2296-2308; framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, L. G. et al., J. Immunol. 151 (1993) 2623-2632); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro, J. C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 (1997) 10678-10684 and Rosok, M. J. et al., J. Biol. Chem. 271 (19969 22611-22618).

Human Antibodies

In certain embodiments, an antibody analyzed in the method as reported herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk, M. A. and van de Winkel, J. G., Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opin. Immunol. 20 (2008) 450-459.

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125. See also, e.g., U.S. Pat. No. 6,075,181 and U.S. Pat. No. 6,150,584 describing XENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HuMab® technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology, and US 2007/0061900, describing VelociMouse® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor, D., J. Immunol. 133 (1984) 3001-3005; Brodeur, B. R. et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; and Boerner, P. et al., J. Immunol. 147 (1991) 86-95) Human antibodies generated via human B-cell hybridoma technology are also described in Li, J. et al., Proc. Natl. Acad. Sci. USA 103 (2006) 3557-3562. Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, J., Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers, H. P. and Brandlein, S., Histology and Histopathology 20 (2005) 927-937 and Vollmers, H. P. and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

Library-Derived Antibodies

Antibodies analyzed in the methods as reported herein may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom, H. R. et al., Methods in Molecular Biology 178 (2001) 1-37 and further described, e.g., in the McCafferty, J. et al., Nature 348 (1990) 552-554; Clackson, T. et al., Nature 352 (1991) 624-628; Marks, J. D. et al., J. Mol. Biol. 222 (1992) 581-597; Marks, J. D. and Bradbury, A., Methods in Molecular Biology 248 (2003) 161-175; Sidhu, S. S. et al., J. Mol. Biol. 338 (2004) 299-310; Lee, C. V. et al., J. Mol. Biol. 340 (2004) 1073-1093; Fellouse, F. A., Proc. Natl. Acad. Sci. USA 101 (2004) 12467-12472; and Lee, C. V. et al., J. Immunol. Methods 284 (2004) 119-132.

In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter, G. et al., Ann. Rev. Immunol. 12 (1994) 433-455. Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths, A. D. et al., EMBO J. 12 (1993) 725-734. Finally, naive libraries can also be made synthetically by cloning non-rearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol. Biol. 227 (1992) 381-388. Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US 2005/0079574, US 2005/0119455, US 2005/0266000, US 2007/0117126, US 2007/0160598, US 2007/0237764, US 2007/0292936, and US 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

Multispecific Antibodies

Engineered proteins, such as bi- or multispecific antibodies capable of binding two or more antigens are known in the art. Such multispecific binding proteins can be generated using cell fusion, chemical conjugation, or recombinant DNA techniques.

In certain embodiments, an antibody analyzed in the methods as reported herein is a multispecific antibody, e.g. at least a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. Multispecific antibodies can be prepared as full length antibodies.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny, S. A. et al., J. Immunol. 148 (1992) 1547-1553; using “diabody” technology for making bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (sFv) dimers (see, e.g. Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies as described, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included herein (see, e.g. US 2006/0025576).

The antibody or fragment herein also includes a “Dual Acting Fab” or “DAF” comprising an antigen binding site that binds to [[PRO]] as well as another, different antigen (see, US 2008/0069820, for example).

The antibody or fragment herein also includes multispecific antibodies described in

WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792, and WO 2010/145793.

A wide variety of recombinant multispecific antibody formats have been developed in the recent past, e.g. tetravalent bispecific antibodies by fusion of, e.g. an IgG antibody format and single chain domains (see e.g. Coloma, M. J., et. al., Nature Biotech. 15 (1997) 159-163; WO 2001/077342; and Morrison, S. L., Nature Biotech. 25 (2007) 1233-1234.

Also several other new formats wherein the antibody core structure (IgA, IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- or tetrabodies, minibodies, several single chain formats (scFv, Bis-scFv), which are capable of binding two or more antigens, have been developed (Holliger, P., et. al, Nature Biotech. 23 (2005) 1126-1136; Fischer, N., and Leger, O., Pathobiology 74 (2007) 3-14; Shen, J., et. al., J. Immunol. Methods 318 (2007) 65-74; Wu, C., et al., Nature Biotech. 25 (2007) 1290-1297).

All such formats use linkers either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fuse e.g. two Fab fragments or scFv (Fischer, N., and Léger, O., Pathobiology 74 (2007) 3-14). While it is obvious that linkers have advantages for the engineering of multispecific antibodies, they may also cause problems in therapeutic settings. Indeed, these foreign peptides might elicit an immune response against the linker itself or the junction between the protein and the linker. Furthermore, the flexible nature of these peptides makes them more prone to proteolytic cleavage, potentially leading to poor antibody stability, aggregation and increased immunogenicity. In addition one may want to retain effector functions, such as e.g. complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC), which are mediated through the Fc-part by maintaining a high degree of similarity to naturally occurring antibodies.

Thus, ideally, one should aim at developing multispecific antibodies that are very similar in general structure to naturally occurring antibodies (like IgA, IgD, IgE, IgG or IgM) with minimal deviation from human sequences.

In one approach bispecific antibodies that are very similar to natural antibodies have been produced using the quadroma technology (see Milstein, C., and Cuello, A. C., Nature 305 (1983) 537-540) based on the somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired specificities of the bispecific antibody. Because of the random pairing of two different antibody heavy and light chains within the resulting hybrid-hybridoma (or quadroma) cell line, up to ten different antibody species are generated of which only one is the desired, functional bispecific antibody. Due to the presence of mispaired byproducts, and significantly reduced production yields, sophisticated purification procedures are required (see e.g. Morrison, S. L., Nature Biotech. 25 (2007) 1233-1234). In general the same problem of mispaired by-products remains if recombinant expression techniques are used.

An approach to circumvent the problem of mispaired byproducts, which is known as ‘knobs-into-holes’, aims at forcing the pairing of two different antibody heavy chains by introducing mutations into the CH3 domains to modify the contact interface. On one chain bulky amino acids were replaced by amino acids with short side chains to create a ‘hole’. Conversely, amino acids with large side chains were introduced into the other CH3 domain, to create a ‘knob’. By co-expressing these two heavy chains (and two identical light chains, which have to be appropriate for both heavy chains), high yields of heterodimer formation (knob-hole′) versus homodimer formation (hole-hole′ or ‘knob-knob’) was observed (Ridgway, J. B., et al., Protein Eng. 9 (1996) 617-621; and WO 96/027011). The percentage of heterodimer could be further increased by remodeling the interaction surfaces of the two CH3 domains using a phage display approach and the introduction of a disulfide bridge to stabilize the heterodimers (Merchant, A. M., et al., Nature Biotech. 16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35). New approaches for the knobs-into-holes technology are described in e.g. in EP 1 870 459 A1. Although this format appears very attractive, no data describing progression towards the clinic are currently available. One important constraint of this strategy is that the light chains of the two parent antibodies have to be identical to prevent mispairing and formation of inactive molecules. Thus this technique is not appropriate as a basis for easily developing recombinant, tri- or tetraspecific antibodies against three or four antigens starting from two antibodies against the first and the second antigen, as either the heavy chains of these antibodies and/or the identical light chains have to be optimized first and then further antigen binding peptides against the third and fourth antigen have to be added.

WO 2006/093794 relates to heterodimeric protein binding compositions. WO 99/37791 describes multipurpose antibody derivatives. Morrison, S. L., et al., J. Immunol. 160 (1998) 2802-2808 refers to the influence of variable region domain exchange on the functional properties of IgG.

WO 2013/02362 relates to heterodimerized polypeptides. WO 2013/12733 relates to polypeptides comprising heterodimeric Fc regions. WO 2012/131555 relates to engineered hetero-dimeric immunoglobulins. EP 2647707 relates to engineered hetero-dimeric immunoglobulins.

WO 2013/026835 relates to bispecific, Fc free antibodies with a domain crossover. WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191 relate to bivalent, bispecific IgG antibodies with a domain crossover.

The multispecific antibodies with VH/VL replacement/exchange in one binding to prevent light chain mispairing (CrossMabVH-VL) which are described in WO2009/080252, (see also Schaefer, W. et al, PNAS, 108 (2011) 11187-1191) clearly reduce the byproducts caused by the mismatch of a light chain against a first antigen with the wrong heavy chain against the second antigen (compared to approaches without such domain exchange). However their preparation is not completely free of side products. The main side product is based on a Bence-Jones-type interaction (see also Schaefer, W. et al, PNAS, 108 (2011) 11187-1191; in Fig. S1I of the Supplement).

Bispecific Antibodies

In one embodiment the multispecific antibody is a bispecific antibody.

One aspect as reported herein is a bivalent, bispecific antibody comprising

-   -   a) a first light chain and a first heavy chain of an antibody         specifically binding to a first antigen, and     -   b) a second light chain and a second heavy chain of an antibody         specifically binding to a second antigen, wherein the variable         domains VL and VH of the second light chain and the second heavy         chain are replaced by each other,     -   wherein the first and the second antigen are different antigens.

The antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain under a) are isolated chains.

In the antibody under b)

-   -   within the light chain         -   the variable light chain domain VL is replaced by the             variable heavy chain domain VH of said antibody,     -   and     -   within the heavy chain         -   the variable heavy chain domain VH is replaced by the             variable light chain domain VL of said antibody.

In one embodiment

-   -   i) in the constant domain CL of the first light chain under a)         the amino acid at position 124 (numbering according to Kabat) is         substituted by a positively charged amino acid, and wherein in         the constant domain CH1 of the first heavy chain under a) the         amino acid at position 147 or the amino acid at position 213         (numbering according to Kabat EU index) is substituted by a         negatively charged amino acid,     -   or     -   ii) in the constant domain CL of the second light chain under b)         the amino acid at position 124 (numbering according to Kabat) is         substituted by a positively charged amino acid, and wherein in         the constant domain CH1 of the second heavy chain under b) the         amino acid at position 147 or the amino acid at position 213         (numbering according to Kabat EU index) is substituted by a         negatively charged amino acid.

In one preferred embodiment

-   -   i) in the constant domain CL of the first light chain under a)         the amino acid at position 124 is substituted independently by         lysine (K), arginine (R) or histidine (H) (numbering according         to Kabat) (in one preferred embodiment independently by         lysine (K) or arginine (R)), and wherein in the constant domain         CH1 of the first heavy chain under a) the amino acid at position         147 or the amino acid at position 213 is substituted         independently by glutamic acid (E) or aspartic acid (D)         (numbering according to Kabat EU index),     -   or     -   ii) in the constant domain CL of the second light chain under b)         the amino acid at position 124 is substituted independently by         lysine (K), arginine (R) or histidine (H) (numbering according         to Kabat) (in one preferred embodiment independently by         lysine (K) or arginine (R)), and wherein in the constant domain         CH1 of the second heavy chain under b) the amino acid at         position 147 or the amino acid at position 213 is substituted         independently by glutamic acid (E) or aspartic acid (D)         (numbering according to Kabat EU index).

In one embodiment in the constant domain CL of the second heavy chain the amino acids at position 124 and 123 are substituted by K (numbering according to Kabat EU index).

In one embodiment in the constant domain CH1 of the second light chain the amino acids at position 147 and 213 are substituted by E (numbering according to EU index of Kabat).

In one preferred embodiment in the constant domain CL of the first light chain the amino acids at position 124 and 123 are substituted by K, and in the constant domain CH1 of the first heavy chain the amino acids at position 147 and 213 are substituted by E (numbering according to Kabat EU index).

In one embodiment in the constant domain CL of the second heavy chain the amino acids at position 124 and 123 are substituted by K, and wherein in the constant domain CH1 of the second light chain the amino acids at position 147 and 213 are substituted by E, and in the variable domain VL of the first light chain the amino acid at position 38 is substituted by K, in the variable domain VH of the first heavy chain the amino acid at position 39 is substituted by E, in the variable domain VL of the second heavy chain the amino acid at position 38 is substituted by K, and in the variable domain VH of the second light chain the amino acid at position 39 is substituted by E (numbering according to Kabat EU index).

One aspect as reported herein is a bivalent, bispecific antibody comprising

-   -   a) a first light chain and a first heavy chain of an antibody         specifically binding to a first antigen, and     -   b) a second light chain and a second heavy chain of an antibody         specifically binding to a second antigen, wherein the variable         domains VL and VH of the second light chain and the second heavy         chain are replaced by each other, and wherein the constant         domains CL and CH1 of the second light chain and the second         heavy chain are replaced by each other,     -   wherein the first and the second antigen are different antigens.

The antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain and a) are isolated chains.

In the antibody under b)

-   -   within the light chain         -   the variable light chain domain VL is replaced by the             variable heavy chain domain VH of said antibody, and the             constant light chain domain CL is replaced by the constant             heavy chain domain CH1 of said antibody;     -   and     -   within the heavy chain         -   the variable heavy chain domain VH is replaced by the             variable light chain domain VL of said antibody, and the             constant heavy chain domain CH1 is replaced by the constant             light chain domain CL of said antibody.

One aspect as reported herein is a bivalent, bispecific antibody comprising

-   -   a) a first light chain and a first heavy chain of an antibody         specifically binding to a first antigen, and     -   b) a second light chain and a second heavy chain of an antibody         specifically binding to a second antigen, wherein the constant         domains CL and CH1 of the second light chain and the second         heavy chain are replaced by each other,     -   wherein the first and the second antigen are different antigens.

The antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain under a) are isolated chains.

In the antibody under b)

-   -   within the light chain         -   the constant light chain domain CL is replaced by the             constant heavy chain domain CH1 of said antibody;     -   and within the heavy chain         -   the constant heavy chain domain CH1 is replaced by the             constant light chain domain CL of said antibody.

One aspect as reported herein is a multispecific antibody comprising

-   -   a) a full length antibody specifically binding to a first         antigen and consisting of two antibody heavy chains and two         antibody light chains, and     -   b) one, two, three or four single chain Fab fragments         specifically binding to one to four further antigens (i.e. a         second and/or third and/or fourth and/or fifth antigen,         preferably specifically binding to one further antigen, i.e. a         second antigen),     -   wherein said single chain Fab fragments under b) are fused to         said full length antibody under a) via a peptidic linker at the         C- or N-terminus of the heavy or light chain of said full length         antibody,     -   wherein the first and the second antigen are different antigens.

In one embodiment one or two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C-terminus of the heavy or light chains of said full length antibody.

In one embodiment one or two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C-terminus of the heavy chains of said full length antibody.

In one embodiment one or two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C-terminus of the light chains of said full length antibody.

In one embodiment two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C-terminus of each heavy or light chain of said full length antibody.

In one embodiment two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C-terminus of each heavy chain of said full length antibody.

In one embodiment two identical single chain Fab fragments binding to a second antigen are fused to said full length antibody via a peptidic linker at the C-terminus of each light chain of said full length antibody.

One aspect as reported herein is a trivalent, bispecific antibody comprising

-   -   a) a full length antibody specifically binding to a first         antigen and consisting of two antibody heavy chains and two         antibody light chains,     -   b) a first polypeptide consisting of         -   ba) an antibody heavy chain variable domain (VH),         -   or         -   bb) an antibody heavy chain variable domain (VH) and an             antibody constant domain 1 (CH1),     -    wherein said first polypeptide is fused with the N-terminus of         its VH domain via a peptidic linker to the C-terminus of one of         the two heavy chains of said full length antibody,     -   c) a second polypeptide consisting of         -   ca) an antibody light chain variable domain (VL),         -   or         -   cb) an antibody light chain variable domain (VL) and an             antibody light chain constant domain (CL),     -    wherein said second polypeptide is fused with the N-terminus of         the VL domain via a peptidic linker to the C-terminus of the         other of the two heavy chains of said full length antibody,     -   and     -   wherein the antibody heavy chain variable domain (VH) of the         first polypeptide and the antibody light chain variable domain         (VL) of the second polypeptide together form an antigen-binding         site specifically binding to a second antigen,     -   and     -   wherein the first and the second antigen are different antigens.

In one embodiment the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) are linked and stabilized via an interchain disulfide bridge by introduction of a disulfide bond between the following positions:

-   -   i) heavy chain variable domain position 44 to light chain         variable domain position 100, or     -   ii) heavy chain variable domain position 105 to light chain         variable domain position 43, or     -   iii) heavy chain variable domain position 101 to light chain         variable domain position 100 (numbering always according to         Kabat EU index).

Techniques to introduce unnatural disulfide bridges for stabilization are described e.g. in WO 94/029350, Rajagopal, V., et al., Prot. Eng. (1997) 1453-59; Kobayashi, H., et al., Nuclear Medicine & Biology, Vol. 25, (1998) 387-393; or Schmidt, M., et al., Oncogene (1999) 18 1711-1721. In one embodiment the optional disulfide bond between the variable domains of the polypeptides under b) and c) is between heavy chain variable domain position 44 and light chain variable domain position 100. In one embodiment the optional disulfide bond between the variable domains of the polypeptides under b) and c) is between heavy chain variable domain position 105 and light chain variable domain position 43. (numbering always according to EU index of Kabat) In one embodiment a trivalent, bispecific antibody without said optional disulfide stabilization between the variable domains VH and VL of the single chain Fab fragments is preferred.

One aspect as reported herein is a trispecific or tetraspecific antibody, comprising

-   -   a) a first light chain and a first heavy chain of a full length         antibody which specifically binds to a first antigen, and     -   b) a second (modified) light chain and a second (modified) heavy         chain of a full length antibody which specifically binds to a         second antigen, wherein the variable domains VL and VH are         replaced by each other, and/or wherein the constant domains CL         and CH1 are replaced by each other, and     -   c) wherein one to four antigen binding peptides which         specifically bind to one or two further antigens (i.e. to a         third and/or fourth antigen) are fused via a peptidic linker to         the C- or N-terminus of the light chains or heavy chains of a)         and/or b),     -   wherein the first and the second antigen are different antigens.

The antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain and a) are isolated chains.

In one embodiment the trispecific or tetraspecific antibody comprises under c) one or two antigen binding peptides which specifically bind to one or two further antigens.

In one embodiment the antigen binding peptides are selected from the group of a scFv fragment and a scFab fragment.

In one embodiment the antigen binding peptides are scFv fragments.

In one embodiment the antigen binding peptides are scFab fragments.

In one embodiment the antigen binding peptides are fused to the C-terminus of the heavy chains of a) and/or b).

In one embodiment the trispecific or tetraspecific antibody comprises under c) one or two antigen binding peptides which specifically bind to one further antigen.

In one embodiment the trispecific or tetraspecific antibody comprises under c) two identical antigen binding peptides which specifically bind to a third antigen. In one preferred embodiment such two identical antigen binding peptides are fused both via the same peptidic linker to the C-terminus of the heavy chains of a) and b). In one preferred embodiment the two identical antigen binding peptides are either a scFv fragment or a scFab fragment.

In one embodiment the trispecific or tetraspecific antibody comprises under c) two antigen binding peptides which specifically bind to a third and a fourth antigen. In one embodiment said two antigen binding peptides are fused both via the same peptide connector to the C-terminus of the heavy chains of a) and b). In one preferred embodiment said two antigen binding peptides are either a scFv fragment or a scFab fragment.

One aspect as reported herein is a bispecific, tetravalent antibody comprising

-   -   a) two light chains and two heavy chains of an antibody, which         specifically bind to a first antigen (and comprise two Fab         fragments),     -   b) two additional Fab fragments of an antibody, which         specifically bind to a second antigen, wherein said additional         Fab fragments are fused both via a peptidic linker either to the         C- or N-termini of the heavy chains of a),     -   and     -   wherein in the Fab fragments the following modifications were         performed         -   i) in both Fab fragments of a), or in both Fab fragments of             b), the variable domains VL and VH are replaced by each             other, and/or the constant domains CL and CH1 are replaced             by each other,         -   or         -   ii) in both Fab fragments of a) the variable domains VL and             VH are replaced by each other, and the constant domains CL             and CH1 are replaced by each other,             -   and             -   in both Fab fragments of b) the variable domains VL and                 VH are replaced by each other, or the constant domains                 CL and CH1 are replaced by each other,         -   or         -   iii) in both Fab fragments of a) the variable domains VL and             VH are replaced by each other, or the constant domains CL             and CH1 are replaced by each other,             -   and             -   in both Fab fragments of b) the variable domains VL and                 VH are replaced by each other, and the constant domains                 CL and CH1 are replaced by each other,         -   or         -   iv) in both Fab fragments of a) the variable domains VL and             VH are replaced by each other, and in both Fab fragments             of b) the constant domains CL and CH1 are replaced by each             other,         -   or         -   v) in both Fab fragments of a) the constant domains CL and             CH1 are replaced by each other, and in both Fab fragments             of b) the variable domains VL and VH are replaced by each             other,     -   wherein the first and the second antigen are different antigens.

In one embodiment said additional Fab fragments are fused both via a peptidic linker either to the C-termini of the heavy chains of a), or to the N-termini of the heavy chains of a).

In one embodiment said additional Fab fragments are fused both via a peptidic linker either to the C-termini of the heavy chains of a).

In one embodiment said additional Fab fragments are fused both via a peptide connector to the N-termini of the heavy chains of a).

In one embodiment in the Fab fragments the following modifications are performed:

-   -   i) in both Fab fragments of a), or in both Fab fragments of b),         the variable domains VL and VH are replaced by each other,         -   and/or         -   the constant domains CL and CH1 are replaced by each other.

In one embodiment in the Fab fragments the following modifications are performed:

-   -   i) in both Fab fragments of a) the variable domains VL and VH         are replaced by each other,         -   and/or         -   the constant domains CL and CH1 are replaced by each other.

In one embodiment in the Fab fragments the following modifications are performed:

-   -   i) in both Fab fragments of a) the constant domains CL and CH1         are replaced by each other.

In one embodiment in the Fab fragments the following modifications are performed:

-   -   i) in both Fab fragments of b) the variable domains VL and VH         are replaced by each other,         -   and/or         -   the constant domains CL and CH1 are replaced by each other.

In one embodiment in the Fab fragments the following modifications are performed:

-   -   i) in both Fab fragments of b) the constant domains CL and CH1         are replaced by each other.

One aspect as reported herein is a bispecific, tetravalent antibody comprising:

-   -   a) a (modified) heavy chain of a first antibody, which         specifically binds to a first antigen and comprises a first         VH—CH1 domain pair, wherein to the C-terminus of said heavy         chain the N-terminus of a second VH—CH1 domain pair of said         first antibody is fused via a peptidic linker,     -   b) two light chains of said first antibody of a),     -   c) a (modified) heavy chain of a second antibody, which         specifically binds to a second antigen and comprises a first         VH-CL domain pair, wherein to the C-terminus of said heavy chain         the N-terminus of a second VH-CL domain pair of said second         antibody is fused via a peptidic linker, and     -   d) two (modified) light chains of said second antibody of c),         each comprising a CL-CH1 domain pair,     -   wherein the first and the second antigen are different antigens.

One aspect as reported herein is a bispecific antibody comprising

-   -   a) the heavy chain and the light chain of a first full length         antibody that specifically binds to a first antigen, and     -   b) the heavy chain and the light chain of a second full length         antibody that specifically binds to a second antigen, wherein         the N-terminus of the heavy chain is connected to the C-terminus         of the light chain via a peptidic linker,     -   wherein the first and the second antigen are different.

The antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain are isolated chains.

One aspect as reported herein is a bispecific antibody comprising

-   -   a) a full length antibody specifically binding to a first         antigen and consisting of two antibody heavy chains and two         antibody light chains, and     -   b) an Fv fragment specifically binding to a second antigen         comprising a VH² domain and a VL² domain, wherein both domains         are connected to each other via a disulfide bridge,     -   wherein only either the VH² domain or the VL² domain is fused         via a peptidic linker to the heavy or light chain of the full         length antibody specifically binding to a first antigen,     -   wherein the first and the second antigen are different antigens.

In the bispecific the heavy chains and the light chains under a) are isolated chains.

In one embodiment the other of the VH² domain or the VL² domain is not fused via a peptide linker to the heavy or light chain of the full length antibody specifically binding to a first antigen.

In all aspects as reported herein the first light chain comprises a VL domain and a CL domain and the first heavy chain comprises a VH domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain.

In one embodiment of all aspects the antibody as reported herein is a multispecific antibody, which requires heterodimerization of at least two heavy chain polypeptides.

Several approaches for CH3-modifications in order to support heterodimerization have been described, for example in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291, which are herein included by reference. Typically, in the approaches known in the art, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are both engineered in a complementary manner so that the heavy chain comprising one engineered CH3 domain can no longer homodimerize with another heavy chain of the same structure (e.g. a CH3-engineered first heavy chain can no longer homodimerize with another CH3-engineered first heavy chain; and a CH3-engineered second heavy chain can no longer homodimerize with another CH3-engineered second heavy chain). Thereby the heavy chain comprising one engineered CH3 domain is forced to heterodimerize with another heavy chain comprising the CH3 domain, which is engineered in a complementary manner. For this embodiment of the invention, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are engineered in a complementary manner by amino acid substitutions, such that the first heavy chain and the second heavy chain are forced to heterodimerize, whereas the first heavy chain and the second heavy chain can no longer homodimerize (e.g. for steric reasons).

The different approaches for supporting heavy chain heterodimerization known in the art, that were cited and included above, are contemplated as different alternatives used in a multispecific antibody according to the invention, which comprises a “non-crossed Fab region” derived from a first antibody, which specifically binds to a first antigen, and a “crossed Fab region” derived from a second antibody, which specifically binds to a second antigen, in combination with the particular amino acid substitutions described above for the invention.

The CH3 domains of the multispecific antibody as reported herein can be altered by the “knob-into-holes” technology which is described in detail with several examples in e.g. WO 96/027011, Ridgway, J. B., et al., Protein Eng. 9 (1996) 617-621; and Merchant, A. M., et al., Nat. Biotechnol. 16 (1998) 677-681. In this method the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be the “knob”, while the other is the “hole”. The introduction of a disulfide bridge further stabilizes the heterodimers (Merchant, A. M., et al., Nature Biotech. 16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

In one preferred embodiment the multispecific antibody as reported herein comprises a T366W mutation in the CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutations in the CH3 domain of the “hole-chain” (numbering according to Kabat EU index). An additional interchain disulfide bridge between the CH3 domains can also be used (Merchant, A. M., et al., Nature Biotech. 16 (1998) 677-681) e.g. by introducing a Y349C mutation into the CH3 domain of the “knobs chain” and a E356C mutation or a S354C mutation into the CH3 domain of the “hole chain”. Thus in a another preferred embodiment, the multispecific antibody as reported herein comprises the Y349C and T366W mutations in one of the two CH3 domains and the E356C, T366S, L368A and Y407V mutations in the other of the two CH3 domains or the multispecific antibody as reported herein comprises the Y349C and T366W mutations in one of the two CH3 domains and the S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain forming a interchain disulfide bridge) (numbering according to Kabat EU index).

But also other knobs-in-holes technologies as described by EP 1 870 459A1, can be used alternatively or additionally. In one embodiment the multispecific antibody as reported herein comprises the R409D and K370E mutations in the CH3 domain of the “knobs chain” and the D399K and E357K mutations in the CH3 domain of the “hole-chain” (numbering according to Kabat EU index).

In one embodiment the multispecific antibody as reported herein comprises a T366W mutation in the CH3 domain of the “knobs chain” and the T366S, L368A and Y407V mutations in the CH3 domain of the “hole chain” and additionally the R409D and K370E mutations in the CH3 domain of the “knobs chain” and the D399K and E357K mutations in the CH3 domain of the “hole chain” (numbering according to the Kabat EU index).

In one embodiment the multispecific antibody as reported herein comprises the Y349C and T366W mutations in one of the two CH3 domains and the S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains, or the multispecific antibody as reported herein comprises the Y349C and T366W mutations in one of the two CH3 domains and the S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains and additionally the R409D and K370E mutations in the CH3 domain of the “knobs chain” and the D399K and E357K mutations in the CH3 domain of the “hole chain” (numbering according to the Kabat EU index).

Apart from the “knob-into-hole technology” other techniques for modifying the CH3 domains of the heavy chains of a multispecific antibody to enforce heterodimerization are known in the art. These technologies, especially the ones described in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954 and WO 2013/096291 are contemplated herein as alternatives to the “knob-into-hole technology” in combination with a multispecific antibody as reported herein.

In one embodiment of a multispecific antibody as reported herein the approach described in EP 1870459 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3-domain-interface between both, the first and the second heavy chain.

Accordingly, this embodiment relates to a multispecific antibody as reported herein, wherein in the tertiary structure of the antibody the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain form an interface that is located between the respective antibody CH3 domains, wherein the respective amino acid sequences of the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain each comprise a set of amino acids that is located within said interface in the tertiary structure of the antibody, wherein from the set of amino acids that is located in the interface in the CH3 domain of one heavy chain a first amino acid is substituted by a positively charged amino acid and from the set of amino acids that is located in the interface in the CH3 domain of the other heavy chain a second amino acid is substituted by a negatively charged amino acid. The multispecific antibody according to this embodiment is herein also referred to as “CH3(+/−)-engineered multispecific antibody” (wherein the abbreviation “+/−” stands for the oppositely charged amino acids that were introduced in the respective CH3 domains).

In one embodiment of said CH3(+/−)-engineered multispecific antibody as reported herein the positively charged amino acid is selected from K, R and H, and the negatively charged amino acid is selected from E or D.

In one embodiment of said CH3(+/−)-engineered multispecific antibody as reported herein the positively charged amino acid is selected from K and R, and the negatively charged amino acid is selected from E or D.

In one embodiment of said CH3(+/−)-engineered multispecific antibody as reported herein the positively charged amino acid is K, and the negatively charged amino acid is E.

In one embodiment of said CH3(+/−)-engineered multispecific antibody as reported herein in the CH3 domain of one heavy chain the amino acid R at position 409 is substituted by D and the amino acid K at position is substituted by E, and in the CH3 domain of the other heavy chain the amino acid D at position 399 is substituted by K and the amino acid E at position 357 is substituted by K (numbering according to Kabat EU index).

In one embodiment of a multispecific antibody as reported herein the approach described in WO2013/157953 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. In one embodiment of said multispecific antibody as reported herein, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by K, and in the CH3 domain of the other heavy chain the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index). In another embodiment of said multispecific antibody as reported herein, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by K and the amino acid L at position 351 is substituted by K, and in the CH3 domain of the other heavy chain the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index).

In another embodiment of said multispecific antibody as reported herein, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by K and the amino acid L at position 351 is substituted by K, and in the CH3 domain of the other heavy chain the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index). Additionally at least one of the following substitutions is comprised in the CH3 domain of the other heavy chain: the amino acid Y at position 349 is substituted by E, the amino acid Y at position 349 is substituted by D and the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index). In one embodiment the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index).

In one embodiment of a multispecific antibody as reported herein the approach described in WO2012/058768 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. In one embodiment of said multispecific antibody as reported herein, in the CH3 domain of one heavy chain the amino acid L at position 351 is substituted by Y and the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain the amino acid T at position 366 is substituted by A and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index). In another embodiment, in addition to the aforementioned substitutions, in the CH3 domain of the other heavy chain at least one of the amino acids at positions 411 (originally T), 399 (originally D), 400 (originally S), 405 (originally F), 390 (originally N) and 392 (originally K) is substituted (numbering according to Kabat EU index). Preferred substitutions are:

-   -   substituting the amino acid T at position 411 by an amino acid         selected from N, R, Q, K, D, E and W (numbering according to         Kabat EU index),     -   substituting the amino acid D at position 399 by an amino acid         selected from R, W, Y, and K (numbering according to Kabat EU         index),     -   substituting the amino acid S at position 400 by an amino acid         selected from E, D, R and K (numbering according to Kabat EU         index),     -   substituting the amino acid F at position 405 by an amino acid         selected from I, M, T, S, V and W (numbering according to Kabat         EU index;     -   substituting the amino acid N at position 390 by an amino acid         selected from R, K and D (numbering according to Kabat EU index;         and     -   substituting the amino acid K at position 392 by an amino acid         selected from V, M, R, L, F and E (numbering according to Kabat         EU index).

In another embodiment of said multispecific antibody as reported herein (engineered according to WO2012/058768), in the CH3 domain of one heavy chain the amino acid L at position 351 is substituted by Y and the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain the amino acid T at position 366 is substituted by V and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index). In another embodiment of said multispecific antibody as reported herein, in the CH3 domain of one heavy chain the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain the amino acid T at position 366 is substituted by A and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index). In said last aforementioned embodiment, in the CH3 domain of said other heavy chain the amino acid K at position 392 is substituted by E, the amino acid T at position 411 is substituted by E, the amino acid D at position 399 is substituted by R and the amino acid S at position 400 is substituted by R (numbering according to Kabat EU index).

In one embodiment of a multispecific antibody as reported herein the approach described in WO 2011/143545 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. In one embodiment of said multispecific antibody as reported herein, amino acid modifications in the CH3 domains of both heavy chains are introduced at positions 368 and/or 409 (numbering according to Kabat EU index).

In one embodiment of a multispecific antibody as reported herein the approach described in WO 2011/090762 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. WO 2011/090762 relates to amino acid modifications according to the “knob-into-hole” technology. In one embodiment of said CH3(KiH)-engineered multispecific antibody as reported herein, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by W, and in the CH3 domain of the other heavy chain the amino acid Y at position 407 is substituted by A (numbering according to Kabat EU index). In another embodiment of said CH3(KiH)-engineered multispecific antibody as reported herein, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by Y, and in the CH3 domain of the other heavy chain the amino acid Y at position 407 is substituted by T (numbering according to Kabat EU index).

In one embodiment of a multispecific antibody as reported herein, which is of IgG2 isotype, the approach described in WO 2011/090762 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody.

In one embodiment of a multispecific antibody as reported herein, the approach described in WO 2009/089004 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. In one embodiment of said multispecific antibody as reported herein, in the CH3 domain of one heavy chain the amino acid K or N at position 392 is substituted by a negatively charged amino acid (in one preferred embodiment by E or D, in one preferred embodiment by D), and in the CH3 domain of the other heavy chain the amino acid D at position 399 the amino acid E or D at position 356 or the amino acid E at position 357 is substituted by a positively charged amino acid (in one preferred embodiment K or R, in one preferred embodiment by K, in one preferred embodiment the amino acids at positions 399 or 356 are substituted by K) (numbering according to Kabat EU index). In one further embodiment, in addition to the aforementioned substitutions, in the CH3 domain of the one heavy chain the amino acid K or R at position 409 is substituted by a negatively charged amino acid (in one preferred embodiment by E or D, in one preferred embodiment by D) (numbering according to Kabat EU index). In one even further embodiment, in addition to or alternatively to the aforementioned substitutions, in the CH3 domain of the one heavy chain the amino acid K at position 439 and/or the amino acid K at position 370 is substituted independently from each other by a negatively charged amino acid (in one preferred embodiment by E or D, in one preferred embodiment by D) (numbering according to Kabat EU index).

In one embodiment of a multispecific antibody as reported herein, the approach described in WO 2007/147901 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. In one embodiment of said multispecific antibody as reported herein, in the CH3 domain of one heavy chain the amino acid K at position 253 is substituted by E, the amino acid D at position 282 is substituted by K and the amino acid K at position 322 is substituted by D, and in the CH3 domain of the other heavy chain the amino acid D at position 239 is substituted by K, the amino acid E at position 240 is substituted by K and the amino acid K at position 292 is substituted by D (numbering according to Kabat EU index).

In one embodiment of a multispecific antibody as reported herein, the approach described in WO 2007/110205 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody.

In one embodiment of all aspects and embodiments as reported herein the multispecific antibody is a bispecific antibody or a trispecific antibody. In one preferred embodiment of the invention the multispecific antibody is a bispecific antibody.

In one embodiment of all aspects as reported herein, the antibody is a bivalent or trivalent antibody. In one embodiment the antibody is a bivalent antibody.

In one embodiment of all aspects as reported herein, the multispecific antibody has a constant domain structure of an IgG type antibody. In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG1, or of human subclass IgG1 with the mutations L234A and L235A. In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG2. In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG3. In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG4 or, of human subclass IgG4 with the additional mutation S228P. In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG1 or human subclass IgG4. In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG1 with the mutations L234A and L235A (numbering according to Kabat EU index). In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG1 with the mutations L234A, L235A and P329G (numbering according to Kabat EU index). In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG4 with the mutations S228P and L235E (numbering according to Kabat EU index). In one further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that said multispecific antibody is of human subclass IgG4 with the mutations S228P, L235E and P329G (numbering according to Kabat EU index).

In one embodiment of all aspects as reported herein, an antibody comprising a heavy chain including a CH3 domain as specified herein, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat EU index). In one embodiment of all aspects as reported herein, an antibody comprising a heavy chain including a CH3 domain, as specified herein, comprises an additional C-terminal glycine residue (G446, numbering according to Kabat EU index).

In one embodiment the antibody comprises a first Fc-region polypeptide and a second Fc-region polypeptide, and

-   -   wherein     -   i) the first Fc-region polypeptide is a human IgG1 Fc-region         polypeptide and the second Fc-region polypeptide is a human IgG1         Fc-region polypeptide, or     -   ii) the first Fc-region polypeptide is a human IgG1 Fc-region         polypeptide with the mutations L234A, L235A and the second         Fc-region polypeptide is a human IgG1 Fc-region polypeptide with         the mutations L234A, L235A, or     -   iii) the first Fc-region polypeptide is a human IgG1 Fc-region         polypeptide with the mutations L234A, L235A, P329G and the         second Fc-region polypeptide is a human IgG1 Fc-region         polypeptide with the mutations L234A, L235A, P329G, or     -   iv) the first Fc-region polypeptide is a human IgG1 Fc-region         polypeptide with the mutations L234A, L235A, S354C, T366W and         the second Fc-region polypeptide is a human IgG1 Fc-region         polypeptide with the mutations L234A, L235A, Y349C, T366S,         L368A, Y407V, or     -   v) the first Fc-region polypeptide is a human IgG1 Fc-region         polypeptide with the mutations L234A, L235A, P329G, S354C, T366W         and the second Fc-region polypeptide is a human IgG1 Fc-region         polypeptide with the mutations L234A, L235A, P329G, Y349C,         T366S, L368A, Y407V, or     -   vi) the first Fc-region polypeptide is a human IgG4 Fc-region         polypeptide and the second Fc-region polypeptide is a human IgG4         Fc-region polypeptide, or     -   vii) the first Fc-region polypeptide is a human IgG4 Fc-region         polypeptide with the mutations S228P, L235E and the second         Fc-region polypeptide is a human IgG4 Fc-region polypeptide with         the mutations S228P, L235E, or     -   viii) the first Fc-region polypeptide is a human IgG4 Fc-region         polypeptide with the mutations S228P, L235E, P329G and the         second Fc-region polypeptide is a human IgG4 Fc-region         polypeptide with the mutations S228P, L235E, P329G, or     -   ix) the first Fc-region polypeptide is a human IgG4 Fc-region         polypeptide with the mutations S228P, L235E, S354C, T366W and         the second Fc-region polypeptide is a human IgG4 Fc-region         polypeptide with the mutations S228P, L235E, Y349C, T366S,         L368A, Y407V, or     -   x) the first Fc-region polypeptide is a human IgG4 Fc-region         polypeptide with the mutations S228P, L235E, P329G, S354C, T366W         and the second Fc-region polypeptide is a human IgG4 Fc-region         polypeptide with the mutations S228P, L235E, P329G, Y349C,         T366S, L368A, Y407V.

In one embodiment the antibody comprises a first Fc-region polypeptide and a second Fc-region polypeptide, and

-   -   wherein the antibody comprises the combination of mutations         -   i) I253A, H310A, and H435A, or         -   ii) H310A, H433A, and Y436A, or         -   iii) L251D, L314D, and L432D, or         -   iv) combinations of i) to iii)     -   in the first Fc-region polypeptide and in the second Fc-region         polypeptide.

In one embodiment of all aspects as reported herein the antibody as reported herein is an effector silent antibody. In one embodiment of all aspects as reported herein the antibody is an effector silent antibody and does not bind to human FcRn. In one preferred embodiment of all aspects as reported herein is the antibody of the human subclass IgG1 and has the mutations L234A, L235A, P329G, I253A, H310A and H434A in both heavy chains (numbering according to the Kabat index).

Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. The required nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-[[PRO]] antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of an antibody, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237, U.S. Pat. No. 5,789,199, and U.S. Pat. No. 5,840,523. (See also Charlton, K. A., In: Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J. (2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat. No. 5,959,177, U.S. Pat. No. 6,040,498, U.S. Pat. No. 6,420,548, U.S. Pat. No. 7,125,978, and U.S. Pat. No. 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J. (2004), pp. 255-268.

Specific Embodiments

-   1. Use of a limited digestion with a proteolytic enzyme of a     multispecific antibody for the analysis of the multispecific     antibody's light chain pairing. -   2. Use of a limited digestion with a proteolytic enzyme of a     multispecific antibody for the determination of light chain     mispairing in the multispecific antibody. -   3. Use of a limited digestion with a proteolytic enzyme of a     multispecific antibody produced by a recombinant mammalian cell for     the selection of a multispecific antibody producing mammalian cell. -   4. The use according to any one of embodiments 1 to 3, wherein the     proteolytic enzyme is selected from the group consisting of Lys-C,     Asp-N, Arg-C, Glu-C and chymotrypsin. -   5. The use according to embodiment 4, wherein the proteolytic enzyme     is Lys-C. -   6. The use according to any one of embodiments 1 to 5, wherein the     incubating is for 35 to 45 minutes. -   7. The use according to embodiment 6, wherein the incubating is for     about 40 minutes. -   8. The use according to any one of embodiments 1 to 7, wherein the     multispecific antibody that is incubated with the proteolytic enzyme     is a deglycosylated multispecific antibody. -   9. The use according to any one of embodiments 1 to 8, wherein the     weight ratio of antibody to enzyme is about 1:200. -   10. The use according to any one of embodiments 1 to 9, wherein the     multispecific antibody that is incubated with the proteolytic enzyme     has a concentration of from 200 to 600 μg/mL. -   11. The use according to any one of embodiments 1 to 10, wherein the     multispecific antibody is a monoclonal multispecific antibody. -   12. The use according to any one of embodiments 1 to 11, wherein the     multispecific antibody comprises at least two non-peptidically bound     light chains. -   13. The use according to any one of embodiments 1 to 11, wherein the     multispecific antibody comprises at least three non-peptidically     bound polypeptides. -   14. A method for the determination of the light chain pairing in a     multispecific antibody comprising the following steps:     -   a) incubating a sample comprising the multispecific antibody         with a proteolytic enzyme for a limited time,     -   b) identifying the mass of the fragments obtained by the limited         proteolytic digestion in step a) by mass spectrometry, and     -   c) determining from the results in step b) the light chain         pairing of the multispecific antibody. -   15. A method for the determination of light chain mispairing of a     multispecific antibody comprising the following steps:     -   a) incubating a sample comprising the multispecific antibody         with a proteolytic enzyme for a limited time,     -   b) identifying the mass of the fragments obtained by the limited         proteolytic digestion in step a) by mass spectrometry, and     -   c) determining from the results in step b) the light chain         pairing of the multispecific antibody and thereby determining         light chain mispairing. -   16. A method for the selection of a recombinant mammalian cell     producing a multispecific antibody comprising the following steps:     -   a) individually incubating a sample comprising a multispecific         antibody produced by a clonal population of a recombinant         mammalian cell of a multitude of recombinant mammalian cells all         cells of the multitude producing the same multispecific antibody         with a proteolytic enzyme for a limited time,     -   b) identifying the mass of the fragments obtained by the limited         proteolytic digestion in step a) by mass spectrometry,     -   c) determining from the results in step b) the presence of light         chain mispairing of the multispecific antibody for each clonal         cell population, and     -   d) selecting based on the results in step c) a recombinant cell         producing a multispecific antibody. -   17. The method according to any one of embodiments 14 to 16, wherein     the proteolytic enzyme is selected from the group consisting of     Lys-C, Asp-N, Arg-C, Glu-C and chymotrypsin. -   18. The method according to embodiment 17, wherein the proteolytic     enzyme is Lys-C. -   19. The method according to any one of embodiments 14 to 18, wherein     the incubating is for 35 to 45 minutes. -   20. The method according to embodiment 19, wherein the incubating is     for about 40 minutes. -   21. The method according to any one of embodiments 14 to 20, wherein     the multispecific antibody that is incubated with the proteolytic     enzyme is a deglycosylated multispecific antibody. -   22. The method according to any one of embodiments 14 to 21, wherein     the weight ratio of antibody to enzyme is about 1:200. -   23. The method according to any one of embodiments 14 to 22, wherein     the multispecific antibody that is incubated with the proteolytic     enzyme has a concentration of from 200 to 600 μg/mL. -   24. The method according to any one of embodiments 14 to 23, wherein     step b) is     -   b) desalting the incubation mixture of step a) and identifying         the mass of the fragments obtained by the limited proteolytic         digestion in step a) by mass spectrometry, -   25. The method according to any one of embodiments 14 to 24, wherein     the multispecific antibody is a monoclonal multispecific antibody. -   26. The method according to any one of embodiments 14 to 25, wherein     the multispecific antibody comprises at least two non-peptidically     bound light chains. -   27. The method according to any one of embodiments 14 to 25, wherein     the multispecific antibody comprises at least three non-peptidically     bound polypeptides.

DESCRIPTION OF THE FIGURES

FIG. 1 Correctly assembled (1) and light-chain mispaired (2) forms of a bivalent bispecific antibody.

FIG. 2 Correctly assembled (1) and some light-chain mispaired (2, 3, 4, 5) forms of a tetravalent bispecific antibody.

FIG. 3 IE of plasmin digested bivalent bispecific antibody; light chain 1 and heavy chain 1 form the first binding site and light chain 2 and heavy chain 2 form the second binding site; the individual pairs would be expected at the following positions: A: light chain 2+heavy chain fragment 1, B: light chain 1+heavy chain 1, C: light chain 2+heavy chain 2, D: light chain 1+heavy chain 2.

FIG. 4 IE of plasmin digested bivalent bispecific antibody; light chain 1 and heavy chain 1 form the first binding site and light chain 2 and heavy chain 2 form the second binding site; the individual pairs would be expected at the following positions: A: light chain 2+heavy chain fragment 1, B: light chain 1+heavy chain 1, C: light chain 2+heavy chain 2, D: light chain 1+heavy chain 2.

FIG. 5 IE of limited Lys-C digested bivalent bispecific antibody; light chain 1 and heavy chain 1 form the first binding site and light chain 2 and heavy chain 2 form the second binding site; the individual pairs would be expected at the following positions: A: light chain 2+heavy chain fragment 1, B: light chain 1+heavy chain 1, C: light chain 2+heavy chain 2, D: light chain 1+heavy chain 2.

FIG. 6 IE of a tetravalent bispecific antibody; A: 3 times light chain 1; B: correctly assemble antibody; C: 3 times light chain 2.

FIG. 7 MS-analysis for Protein A affinity chromatography flow-through of proteolytic digested tetravalent bispecific antibody, ISCID of 0; A: correctly paired Fab, B: mispaired Fab.

FIG. 8 MS-analysis for Protein A affinity chromatography flow-through of proteolytic digested tetravalent bispecific antibody, ISCID of 90; A: correctly paired Fab, B: mispaired Fab.

FIG. 9 MS-analysis for Protein A affinity chromatography eluate of proteolytic digested tetravalent bispecific antibody, ISCID of 0; B: correctly paired Fc-Fab, B: mispaired Fc-Fab.

FIG. 10 MS-analysis for Protein A affinity chromatography eluate of proteolytic digested tetravalent bispecific antibody, ISCID of 90; A: correctly paired Fc-Fab, B: mispaired Fc-Fab.

FIG. 11 EI of a pepsin digested tetravalent bispecific antibody.

The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

EXAMPLES Materials & General Methods

General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Amino acids of antibody chains are numbered and referred to according to the numbering systems according to Kabat (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) as defined above.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The molecular biological reagents were used according to the manufacturer's instructions.

Gene Synthesis

Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The 600-1800 bp long gene segments, which were flanked by singular restriction endonuclease cleavage sites, were assembled by annealing and ligating oligonucleotides including PCR amplification and subsequently cloned via the indicated restriction sites e.g. KpnI/SacI or AscI/PacI into a pPCRScript (Stratagene) based pGA4 cloning vector. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene synthesis fragments were ordered according to given specifications at Geneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH (Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software package version 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was used for sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the described antibodies, variants of expression plasmids for transient expression (e.g. in HEK293 EBNA or HEK293-F) cells based either on a cDNA organization with or without a CMV-Intron A promoter or on a genomic organization with a CMV promoter were applied.

Beside the antibody expression cassette the vectors contained:

-   -   an origin of replication which allows replication of this         plasmid in E. coli, and     -   a β-lactamase gene which confers ampicillin resistance in E.         coli.

The transcription unit of the antibody gene was composed of the following elements:

-   -   unique restriction site(s) at the 5′ end     -   the immediate early enhancer and promoter from the human         cytomegalovirus,     -   followed by the Intron A sequence in the case of the cDNA         organization,     -   a 5′-untranslated region of a human antibody gene,     -   an immunoglobulin heavy chain signal sequence,     -   the human antibody chain (wildtype or with domain exchange)         either as cDNA or as genomic organization with the         immunoglobulin exon-intron organization     -   a 3′ untranslated region with a polyadenylation signal sequence,         and     -   unique restriction site(s) at the 3′ end.

The fusion genes comprising the antibody chains as described below were generated by PCR and/or gene synthesis and assembled by known recombinant methods and techniques by connection of the according nucleic acid segments e.g. using unique restriction sites in the respective vectors. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfections larger quantities of the plasmids were prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques were used as described in Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley & Sons, Inc.

Multispecific antibodies were expressed by transient co-transfection of the respective expression plasmids in adherently growing HEK293-EBNA or in HEK29-F cells growing in suspension as described below.

Transient Transfections in HEK293-EBNA System

Multispecific antibodies were expressed by transient co-transfection of the respective expression plasmids (e.g. encoding the heavy and modified heavy chain, as well as the corresponding light and modified light chain) in adherently growing HEK293-EBNA cells (human embryonic kidney cell line 293 expressing Epstein-Barr-Virus nuclear antigen; American type culture collection deposit number ATCC #CRL-10852, Lot. 959 218) cultivated in DMEM (Dulbecco's modified Eagle's medium, Gibco®) supplemented with 10% Ultra Low IgG FCS (fetal calf serum, Gibco®), 2 mM L-Glutamine (Gibco®), and 250 μg/ml Geneticin (Gibco®). For transfection FuGENE™ 6 Transfection Reagent (Roche Molecular Biochemicals) was used in a ratio of FuGENE™ reagent (μl) to DNA (μg) of 4:1 (ranging from 3:1 to 6:1). Proteins were expressed from the respective plasmids using a molar ratio of (modified and wildtype) light chain and heavy chain encoding plasmids of 1:1 (equimolar) ranging from 1:2 to 2:1, respectively. Cells were fed at day 3 with L-Glutamine ad 4 mM, Glucose [Sigma] and NAA [Gibco®]. Multispecific antibody containing cell culture supernatants were harvested from day 5 to 11 after transfection by centrifugation and stored at −20° C. General information regarding the recombinant expression of human immunoglobulins in e.g. HEK293 cells is given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

Transient Transfections in HEK293-F System

Multispecific antibodies were generated by transient transfection with the respective plasmids (e.g. encoding the heavy and modified heavy chain, as well as the corresponding light and modified light chain) using the HEK293-F system (Invitrogen) according to the manufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyle™ 293 expression medium (Invitrogen) were transfected with a mix of the four expression plasmids and 293Fectin™ or fectin (Invitrogen). For 2 L shake flask (Corning) HEK293-F cells were seeded at a density of 1.0E*6 cells/mL in 600 mL and incubated at 120 rpm, 8% CO2. The day after the cells were transfected at a cell density of ca. 1.5E*6 cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 μg total plasmid DNA (1 μg/mL) encoding the heavy or modified heavy chain, respectively and the corresponding light chain in an equimolar ratio and B) 20 ml Opti-MEM+1.2 mL 293 fectin or fectin (2 μl/mL). According to the glucose consumption glucose solution was added during the course of the fermentation. The supernatant containing the secreted antibody was harvested after 5-10 days and antibodies were either directly purified from the supernatant or the supernatant was frozen and stored.

Protein Determination

The protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423.

Antibody Concentration Determination in Supernatants

The concentration of antibodies and derivatives in cell culture supernatants was estimated by immunoprecipitation with Protein A Agarose-beads (Roche). 60 μL Protein A Agarose beads were washed three times in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40). Subsequently, 1-15 mL cell culture supernatant was applied to the Protein A Agarose beads pre-equilibrated in TBS-NP40. After incubation for at 1 hour at room temperature the beads were washed on an Ultrafree-MC-filter column (Amicon) once with 0.5 mL TBS-NP40, twice with 0.5 mL 2× phosphate buffered saline (2×PBS, Roche) and briefly four times with 0.5 mL 100 mM Na-citrate pH 5.0. Bound antibody was eluted by addition of 35 μl NuPAGE® LDS Sample Buffer (Invitrogen). Half of the sample was combined with NuPAGE® Sample Reducing Agent or left unreduced, respectively, and heated for 10 min at 70° C. Consequently, 5-30 μl were applied to a 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen) (with MOPS buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE® Antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE) and stained with Coomassie Blue.

The concentration of antibodies and derivatives in cell culture supernatants was quantitatively measured by affinity HPLC chromatography. Briefly, cell culture supernatants containing antibodies and derivatives that bind to Protein A were applied to an Applied Biosystems Poros A/20 column in 200 mM KH2PO4, 100 mM sodium citrate, pH 7.4 and eluted from the matrix with 200 mM NaCl, 100 mM citric acid, pH 2.5 on an Agilent HPLC 1100 system. The eluted protein was quantified by UV absorbance and integration of peak areas. A purified standard IgG1 antibody served as a standard.

Alternatively, the concentration of antibodies and derivatives in cell culture supernatants was measured by Sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Streptavidin A-96 well microtiter plates (Roche) are coated with 100 biotinylated anti-human IgG capture molecule F(ab′)2<h-Fcγ> BI (Dianova) at 0.1 μg/mL for 1 hour at room temperature or alternatively overnight at 4° C. and subsequently washed three times with 200 μL/well PBS, 0.05% Tween (PBST, Sigma). 100 μL/well of a dilution series in PBS (Sigma) of the respective antibody containing cell culture supernatants was added to the wells and incubated for 1-2 hour on a microtiterplate shaker at room temperature. The wells were washed three times with 200 μL/well PBST and bound antibody was detected with 100 μl F(ab′)2<hFcγ>POD (Dianova) at 0.1 μg/mL as the detection antibody for 1-2 hours on a micro-titerplate shaker at room temperature. Unbound detection antibody was washed away three times with 200 μL/well PBST and the bound detection antibody was detected by addition of 100 μL ABTS/well. Determination of absorbance was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).

Protein Purification

Proteins were purified from filtered cell culture supernatants referring to standard protocols. In brief, antibodies were applied to a Protein A Sepharose column (GE healthcare) and washed with PBS. Elution of antibodies was achieved at pH 2.8 followed by immediate neutralization of the sample. Aggregated protein was separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. Monomeric antibody fractions were pooled, concentrated (if required) using e.g., a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen and stored at −20° C. or −80° C. Part of the samples were provided for subsequent protein analytics and analytical characterization e.g. by SDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to the manufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, with NuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels) running buffer was used.

Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) for the determination of the aggregation and oligomeric state of antibodies was performed by HPLC chromatography. Briefly, Protein A purified antibodies were applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH₂PO₄/K₂HPO₄, pH 7.5 on an Agilent HPLC 1100 system or to a Superdex 200 column (GE Healthcare) in 2×PBS on a Dionex HPLC-System. The eluted protein was quantified by UV absorbance and integration of peak areas. BioRad Gel Filtration Standard 151-1901 served as a standard.

Example 1

Method for the Determination of Light Chain Mispairing of a Multispecific Antibody after Limited Proteolytic Digestion

The expected primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the limited LysC digested CrossMabs. Advantageously the antibody has been deglycosylated in advance.

The VH/VL CrossMabs was deglycosylated with N-Glycosidase F in a phosphate or Tris or histidine buffer at 37° C. for up to 17 h at a protein concentration of 1 mg/mL and an antibody: enzyme ratio of 100:1. The limited Lys-C (Roche Diagnostics GmbH, Mannheim, Germany) digestions was performed with 100 μg deglycosylated VH/VL CrossMabs in a Tris buffer pH 8 at 37° C. for 40 min.

Prior to mass spectrometry the samples were desalted via HPLC on a Sephadex G25 column (GE Healthcare).

The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

Example 2 Method for the Determination of Light Chain Mispairing of a Tetravalent Bispecific Antibody

The tetravalent bispecific antibody was deglycosylated with N-Glycosidase F in a phosphate or Tris or histidine buffer at 37° C. for up to 17 h at a protein concentration of 1 mg/ml and an antibody:enzyme ratio of 100:1. The limited Lys-C(Roche Diagnostics GmbH, Mannheim, Germany) digestions was performed with 100 μg deglycosylated tetravalent bispecific antibody in a 100 mM citrate buffer pH 3.7 at 37° C. for 16 hours at a protein concentration of approx. 0.9 mg/mL and an antibody:enzyme ratio of 50:1. 

1.-3. (canceled)
 4. A method for the determination of the light chain pairing in a multispecific antibody comprising the following steps: a) incubating a sample comprising the multispecific antibody with a proteolytic enzyme for a limited time, b) identifying the mass of the fragments obtained by the limited proteolytic digestion in step a) by mass spectrometry, and c) determining from the results in step b) the light chain pairing of the multispecific antibody.
 5. A method for the determination of light chain mispairing of a multispecific antibody comprising the following steps: a) incubating a sample comprising the multispecific antibody with a proteolytic enzyme for a limited time, b) identifying the mass of the fragments obtained by the limited proteolytic digestion in step a) by mass spectrometry, and c) determining from the results in step b) the light chain pairing of the multispecific antibody and thereby determining light chain mispairing.
 6. A method for the selection of a recombinant mammalian cell producing a multispecific antibody comprising the following steps: a) individually incubating a sample comprising a multispecific antibody produced by a clonal population of a recombinant mammalian cell of a multitude of recombinant mammalian cells all cells of the multitude producing the same multispecific antibody with a proteolytic enzyme for a limited time, b) identifying the mass of the fragments obtained by the limited proteolytic digestion in step a) by mass spectrometry, c) determining from the results in step b) the presence of light chain mispairing of the multispecific antibody for each clonal cell population, and d) selecting based on the results in step c) a recombinant cell producing a multispecific antibody.
 7. The method according to claim 4, wherein the proteolytic enzyme is selected from the group consisting of Lys-C, Asp-N, Arg-C, Glu-C and chymotrypsin.
 8. The method according to claim 7, wherein the proteolytic enzyme is Lys-C.
 9. The method according to claim 4, wherein the incubating is for 35 to 45 minutes.
 10. The method according to claim 9, wherein the incubating is for about 40 minutes.
 11. The method according to claim 4, wherein the multispecific antibody that is incubated with the proteolytic enzyme is a deglycosylated multispecific antibody.
 12. The method according to claim 4, wherein the weight ratio of antibody to enzyme is about 1:200.
 13. The method according to claim 4, wherein the multispecific antibody that is incubated with the proteolytic enzyme has a concentration of from 200 to 600 μg/mL.
 14. The method according to claim 4, wherein step b) is b) desalting the incubation mixture of step a) and identifying the mass of the fragments obtained by the limited proteolytic digestion in step a) by mass spectrometry.
 15. The method according to claim 4, wherein the multispecific antibody is a monoclonal multispecific antibody.
 16. The method according to claim 4, wherein the multispecific antibody comprises at least two non-peptidically bound light chains.
 17. The method according to claim 4, wherein the multispecific antibody comprises at least three non-peptidically bound polypeptides. 